Thrombin is an enzyme that is involved in blood coagulation and is indispensable to maintenance and progress of life such as formation of hemostatic thrombus or treatment of injury. Thrombin is normally present in blood in the form of inactive prothrombin but is activated by the coagulation system triggered by, for instance, platelets or damage in tissue cells to convert fibrinogen in a soluble form into insoluble fibrin useful for hemostasis and treatment of injury. A hemostatic utilizing this principle has widely been applied in clinical scenes.
Although thrombin has been used as an active ingredient of a hemostatic, with the conventional hemostatic in the form of liquid, hemostasis by pressing or closing by pressing to a spot of gushing hemorrhage or oozing hemorrhage is not possible and therefore bleeding cannot be stopped.
Aiming at hemostasis of such gushing hemorrhage or oozing hemorrhage, there have been attempts to develop a sheet-type hemostatic where thrombin, fibrinogen and/or a coagulation factor is held on a collagen sponge, a non-woven fabric consisting of alginic acid or polyglycolic acid, or other bioabsorbable supports consisting of gelatin or hydrogel (e.g. see Patent references 1, 2, 3, 4, 5, 6 and 7).
However, most of the sheet hemostatic have not yet been put into practical usage due to problems that they are thick and lack flexibility since, when applied to a closing spot of organs in their dry form, rigidity of supports per se may adversely affect to thereby make it difficult to closely contact with a spot of injury, thus failing to exert sufficient adhesive effect.
A unique product of a sheet-type fibrin adhesive that has been commercially available is one where fibrinogen and bovine thrombin are held on a support consisting of equine collagen (product name: TachoComb/CSL Behling) (e.g. see Patent reference 8). However, this adhesive still has room for improvement in handling and removal of risk factors in view of its thickness (about 3 mm) and inclusion of components derived from animals.
On the other hand, there are several reports on a stabilizing agent to thrombin. For instance, approaches for preserving thrombin with stability has been reported by comprising a salt of an organic carboxylic acid (e.g. see Patent reference 9) or glycerol (e.g. see Patent references 10, 11 and 12) for a liquid preparation, or by comprising a sugar, a basic amino acid and a liquid organic acid (e.g. see Patent reference 13), or gelatin, glycine and a sugar (e.g. see Patent reference 14), or an aliphatic polybasic carboxylic acid and albumin (e.g. see Patent reference 15), or human serum albumin and an amino acid (e.g. see Patent reference 16) for a dry preparation packed in a vial or in an aluminum pouch package. However, in case of a sheet-type preparation, mere stabilization of thrombin cannot attain an object.
When a sheet preparation is used, it is sometimes made round or folded so that it may touch closely a spot of injury. Therefore, for avoiding breakage of a sheet or leakage of thrombin constituent due to forces imposed, it is necessary to improve flexibility or thrombin-holding property of a sheet.
For instance, the thrombin preparation comprising a sugar, a basic amino acid and a liquid organic acid as described above is not suited for a sheet-type preparation since flexibility is lost when the thrombin preparation is held on a sheet. Also, when a non-woven fabric is immersed in a thrombin solution and is simply subject to lyophilization, thrombin is not held on the non-woven fabric. In order to solve the problems, a technique is reported that a non-woven fabric made of chitin as a support is immersed alternately in three solutions, i.e. a solution of an acidic high molecular weight compound such as chondroitin sulfate, alginic acid or hyaluronic acid, a solution of a basic high molecular weight compound such as chitosan, and a thrombin solution, to hold thrombin, followed by drying in vacuum or with ventilation to prepare a sheet-like hemostatic or a sheet-like medicament for injury (e.g. see Patent reference 17). However, with such technique of alternate absorption, even if flexibility of a sheet could be secured, reduction of the activity of thrombin would be unavoidable and handling in production is cumbersome. Accordingly, development of a more efficient process for preparation is desired.
Patent reference 1: Japanese Patent Publication No. 61-59737
Patent reference 2: Japanese Patent Publication No. 05-163157
Patent reference 3: Japanese Patent Publication No. 2002-515300
Patent reference 4: U.S. Pat. No. 4,453,939
Patent reference 5: Japanese Patent Publication No. 9-504719
Patent reference 6: Japanese Patent Publication No. 2000-510357
Patent reference 7: Japanese Patent Publication No. 2001-513368
Patent reference 8: European Patent 0059265
Patent reference 9: Japanese Patent Publication No. 56-39782
Patent reference 10: Japanese Patent Publication No. 62-106028
Patent reference 11: Japanese Patent Publication No. 7-64747
Patent reference 12: Japanese Patent Publication No. 63-192723
Patent reference 13: Japanese Patent Publication No. 2-53732
Patent reference 14: Japanese Patent Publication No. 7-165604
Patent reference 15: Japanese Patent Publication No. 2002-193832
Patent reference 16: Japanese Patent Publication No. 2006-117678
Patent reference 17: Japanese Patent Publication No. 2006-306759